The Development and Evaluation of A Multiplex Real-time PCR Assay For the Detection of ESBL Genes in Urinary Tract Infections - The Development and Evaluation of A Multiplex Real-time PCR Assay For the Detection of ESBL Genes in Urinary Tract Infections - Open Access Pub

Background Overuse of beta-lactam antibiotics has lead to selection for extended-spectrum β-lactamase (ESBL) producing Enterobacteriaceae, a major cause of antibiotic resistant urinary tract infections (UTIs). Standard detection methods are time-consuming, with disputed accuracy. This study describes a novel real-time PCR method to detect CTX-M, SHV, OXA and TEM. Methods 179 Enterobacteriaceae isolates from UTIs were collected from the Leicester Royal Infirmary, UK. A multiplex Plexor？-based real-time PCR assay detected ESBLs using their specific amplicon melting temperature, during each cycle, removing the need for a melt-curve analysis. Validation was achieved by end-point PCR and disk diffusion. Results The method was able to produce rapid and accurate results, achieving a sensitivity and specificity of 94.9% and 72% respectively, and the assay can differentiate between the different ESBL genes, with ease. Conclusions With further investigation, a Plexor？-based assay could form the basis of a high-throughput kit that health services could use to detect ESBLs or other antibiotic resistance genes. Overuse of beta-lactam antibiotics has lead to selection for extended-spectrum β-lactamase (ESBL) producing Enterobacteriaceae, a major cause of antibiotic resistant urinary tract infections (UTIs). Standard detection methods are time-consuming, with disputed accuracy. This study describes a novel real-time PCR method to detect CTX-M, SHV, OXA and TEM. 179 Enterobacteriaceae isolates from UTIs were collected from the Leicester Royal Infirmary, UK. A multiplex Plexor？-based real-time PCR assay detected ESBLs using their specific amplicon melting temperature, during each cycle, removing the need for a melt-curve analysis. Validation was achieved by end-point PCR and disk diffusion. The method was able to produce rapid and accurate results, achieving a sensitivity and specificity of 94.9% and 72% respectively, and the assay can differentiate between the different ESBL genes, with ease. With further investigation, a Plexor？-based assay could form the basis of a high-throughput kit that health services could use to detect ESBLs or other antibiotic resistance genes. DOI10.14302/issn.2690-4721.ijcm-18-2217 Antibiotic resistance is a natural process, whereby bacteria exchange genes via horizontal transfer, however the overuse of antibiotics has caused a permanent selective force for resistance mechanisms. 1 In the O’Neill report in 2016, it was estimated that every year at least 700,000 people die as a result of antibiotic resistant infections. 2 Extended-spectrum